Clinical evidence suggests a strong link between Alzheimer's disease (AD) and cerebral microvascular dysfunction, but it remains unclear whether they contribute independently to dementia, or if AD pathology triggers microvascular disease, or vice versa. In AD, amyloid-[unreadable] (A[unreadable]) peptides aggregate to form neurotoxic oligomers and eventually accumulate as amyloid plaques. Aggregation of A[unreadable] depends on concentration, so events that increase production or decrease clearance of A[unreadable] could be triggers for AD. A major pathway for the removal of A[unreadable] from the brain is through the vasculature, suggesting that microvascular lesions could interfere with A[unreadable] clearance. In addition, vascular lesions can lead to increased reactive oxygen species and to inflammation, which are linked to A[unreadable] increase. We recently developed optical methods to create lesions in individual, specifically-targeted microvessels in rodent cortex. We now propose to test the hypothesis that microvascular lesions initiate or accelerate A[unreadable] accumulation and amyloid plaque formation. To perform these studies, femtosecond laser irradiation will be used to injure a specifically targeted blood vessel, causing the formation of a microhemorrhage and/or of a clot that stops blood flow. We will produce microvascular clots and hemorrhages in individual cortical penetrating arterioles and capillaries in transgenic AD mice that express amyloid precursor protein (Mo/HuAPP695swe) and mutant presenilin1 (PS1-dE9), as well as in age-matched controls. Amyloid plaques will be labeled in vivo with systemic injections of methoxy-X04, a Congo-red derivative. The location of the lesions will be imaged daily over several days with two-photon excited fluorescence microscopy to determine the persistence of the occlusion or hemorrhage and the presence of previously existing and new amyloid plaques. Post-mortem labeling with thioflavin-S and A[unreadable] antibodies will be used to further elucidate the impact of the microvascular lesion on A[unreadable] accumulation and amyloid plaque formation. In Aim 1, we test whether microvascular clots and hemorrhages trigger rapid amyloid plaque formation for lesions at different locations in the vascular tree, and where amyloid plaques form relative to the lesion site. In Aim 2, we determine the time required for amyloid plaques to form following microvascular lesions and whether amyloid plaques that are initiated by microvascular lesions are stable over time. In the final Aim, we investigate how the age of the animal, or the pre-existing plaque burden, influences the seeding of amyloid plaques by microvascular lesions. In preliminary work, we found that microvascular clots led to the formation of new amyloid plaques within one day (3/3 clots in 3 animals, see Fig. 7). New plaques were formed both on the clotted microvessel and in the nearby parenchymal tissue. Nearby vessels of similar diameter that were not lesioned showed no new amyloid plaques. These initial results indicate that a severe decrease in blood flow resulting from the occlusion of a single microvessel can trigger amyloid plaque formation. These data suggest that microvascular clots could play an important role in Alzheimer's disease pathogenesis. PUBLIC HEALTH RELEVANCE: Alzheimer's disease (AD) is the leading cause of dementia in the elderly and although some recent treatments modestly slow progression of disease, there is no cure. Clinical research has shown that vascular health is an important factor in the severity of AD in patients, yet the mechanisms that link vascular dysfunction and AD remain unclear. This work uses a unique combination of optical and biological tools to directly study how microvascular clots and hemorrhages in the brain affect the development of AD and our preliminary results show that microvascular dysfunction may play a role in initiating AD, suggesting that successful AD prevention will depend on treatment of vascular disease.